Researcher(s)
- Eleanor Cook, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Chinese hamster ovary (CHO) cells play a crucial role in the biopharmaceutical industry due to their ability to produce and secrete recombinant proteins, notably monoclonal antibodies, at titers as high as 10 g/L. The high genomic plasticity and inefficient central metabolism of CHO leaves cells susceptible to genomic instability and the accumulation of toxic byproducts, resulting in reduced cell survival and compromised production of valuable proteins. In addition, this instability and inefficiency cause a reduced tolerance to stress conditions encountered during at-scale production, such as high osmolality and ammonia concentrations. To enhance the stress tolerance of CHO cells, dCas9 fusion proteins linked to chromatin remodeling domains (dCas9-CRD) targeting a gene believed to be associated with stress tolerance (Hmox1) were integrated into CHO cells. These dCas9-CRD constructs varied in whether they upregulated or downregulated the expression of the Hmox1 gene. Future research will target other genes believed to be linked with stress resistance, namely the ATF3 and Ier3 genes, as well as combinations of these genes to optimize stress tolerance in CHO cells. A fed-batch experiment was conducted in which automated cell counting, qPCR, and HPLC were used to measure the cell viability, Hmox1 expression, and antibody titer of CHO cells integrated with these constructs over nine days under control, osmolality stress, and ammonia stress conditions. Upregulators of gene expression, notably p300 and TET1, demonstrated superior survival compared to downregulators under stress conditions, suggesting that increased Hmox1 gene activation may aid CHO cells in resisting ammonia and osmolality stress. Integrating these constructs before or during CHO cell line development could optimize the process of developing more stress-tolerant CHO cell lines capable of improved growth in manufacturing scale bioreactors.